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Antimicrobial Agents and Chemotherapy, June 1998, p. 1417-1423, Vol. 42, No. 6
0066-4804/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Pharmacokinetics of Sparfloxacin in the Serum and Vitreous Humor
of Rabbits: Physicochemical Properties That Regulate
Penetration of Quinolone Antimicrobials
Weiguo
Liu,1
Qing
Feng
Liu,1
Ruth
Perkins,1
George
Drusano,1,2
Arnold
Louie,1
Assumpta
Madu,3
Umar
Mian,3,4
Martin
Mayers,3,4 and
Michael H.
Miller1,*
Divisions of
Infectious
Diseases1 and
Clinical
Pharmacology,2 Departments of Medicine and
Pharmacology, Albany Medical College, Albany, and
Department of Ophthalmology, Montefiore Medical Center,
University Hospital for the Albert Einstein College of
Medicine,3 and
Department of
Ophthalmology, Bronx Lebanon Medical Center, Albert Einstein
College of Medicine,4 Bronx, New York
Received 29 May 1997/Returned for modification 11 December
1997/Accepted 19 March 1998
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ABSTRACT |
We have used a recently described animal model to characterize the
ocular pharmacokinetics of sparfloxacin in vitreous humor of uninfected
albino rabbits following systemic administration and direct
intraocular injection. The relationships of lipophilicity, protein binding, and molecular weight to the penetration and
elimination of sparfloxacin were compared to those of
ciprofloxacin, fleroxacin, and ofloxacin. To determine whether
elimination was active, elimination rates following direct injection
with and without probenecid or heat-killed bacteria were compared.
Sparfloxacin concentrations were measured in the serum and vitreous
humor by a biological assay. Protein binding and lipophilicity were
determined, respectively, by ultrafiltration and oil-water
partitioning. Pharmacokinetic parameters were characterized with
RSTRIP, an iterative, nonlinear, weighted,
least-squares-regression program. The relationship between each
independent variable and mean quinolone concentration or elimination
rate in the vitreous humor was determined by multiple linear
regression. The mean concentration of sparfloxacin in the vitreous
humor was 59.4% ± 12.2% of that in serum. Penetration of
sparfloxacin, ciprofloxacin, fleroxacin, and ofloxacin into, and
elimination from, the vitreous humor correlated with lipophilicity (r2 > 0.999). The linear-regression
equation describing this relationship was not improved by including the
inverse of the square root of the molecular weight and/or the degree of
protein binding. Elimination rates for each quinolone were
decreased by the intraocular administration of probenecid. Heat-killed
Staphylococcus epidermidis decreased the rate of
elimination of fleroxacin. Penetration of sparfloxacin into the
noninflamed vitreous humor was greater than that of any quinolone
previously examined. There was an excellent correlation between lipophilicity and vitreous entry or elimination for
sparfloxacin as well as ciprofloxacin, fleroxacin, and ofloxacin. There
are two modes of quinolone translocation into and out of the vitreous humor: diffusion into the eye and both diffusion and carrier-mediated elimination out of the vitreous humor.
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INTRODUCTION |
Bacterial endophthalmitis is a
severe and often blinding condition (2, 22, 48, 52).
While the direct injection of antimicrobials into the vitreous humor is
known to improve visual outcome, the roles of systemic antibiotics are
less well understood (7, 21, 48, 52). Systemically
administered antimicrobials commonly used in the therapy of
endophthalmitis do not penetrate into the noninflamed
vitreous humor (24, 48, 52). Following cataract
surgery, the intravitreal injection of antimicrobial agents in the
therapy of endophthalmitis, which is primarily due to
Staphylococcus epidermidis, is currently considered the
treatment of choice for most patients (24). However, the
potential role of systemically administered agents that exhibit better
penetration into the vitreous humor has not been studied. Moreover,
neither therapy nor prophylaxis of endophthalmitis of
other causes (e.g., posttraumatic and hematogenous) or microbial
etiologies (e.g., Streptococcus pneumoniae,
Bacillus spp., and Pseudomonas aeruginosa) has
been well characterized.
Since accurate pharmacokinetic data have fundamental implications for
outcome studies of animals and humans, we have developed and validated
an animal model in which sequential vitreous humor samples can be
obtained from a small number of rabbits. Based upon the comparison of
pharmacokinetic parameters in single and serially sampled eyes, we have
shown that serial sampling does not alter ocular pharmacokinetic
parameters. By this approach, the pharmacokinetic parameter estimates
from as few as three animals give more accurate data than it is
possible to obtain with more than 20 times this number of animals by
the approach of combining single datum points from different animals
(23, 35, 41, 43, 51). Our method provides more-robust
parameter estimates that permit the characterization of ocular
pharmacokinetics which are difficult to address by the older approach
(23, 35, 40, 41, 43, 51).
Studies in our laboratory (23, 41, 43) and by others
(16, 39) have shown that quinolones penetrate into the
noninflamed vitreous better than beta-lactams, aminoglycosides, or
vancomycin (5, 31, 34, 36, 38, 59, 60). Based primarily upon these penetration data, systemically administered ciprofloxacin has
been used to treat patients with bacterial
endophthalmitis (32). However, the activity
of ciprofloxacin against ocular pathogens, particularly
coagulase-negative staphylococci, is marginal and its penetration is
poor relative to that of fleroxacin (43) or ofloxacin
(51). Sparfloxacin, a recently introduced quinolone antimicrobial (14, 54, 58), is more active against
staphylococci and appears to penetrate into the noninflamed vitreous
better than ciprofloxacin (16, 23, 41). However, the
existing pharmacokinetic data are based upon studies which combine
single samples from different subjects to generate pharmacokinetic
estimates. This method is unreliable when used to describe
pharmacokinetic data in humans (55).
The primary goals of the current study were threefold. We wanted to (i)
characterize the ocular pharmacokinetics of sparfloxacin, (ii) compare
the relationships of protein binding, lipophilicity, and molecular
weight (MW) to the vitreous translocation (entry and elimination)
of sparfloxacin with those of other quinolones, and (iii) determine if
the elimination of these drugs was blocked by probenecid or
heat-killed bacteria.
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MATERIALS AND METHODS |
Animal model.
Adult male, New Zealand White rabbits
(Milbrook Farms, Amherst, Mass.) weighing 2 to 3 kg were used. Animals
were obtained and cared for in accordance with Association for Research
in Vision and Ophthalmology guidelines. The care, anesthesia, and
vitreous sampling methods were similar to those described previously
(43). The animals were anesthetized with an intramuscular
dose of diazepam (2.5 mg) and a subcutaneous dose of urethane (1.62 g/kg of body weight) given approximately 45 min prior to antibiotic
administration. Anesthesia was maintained throughout the sampling
period, with administration of supplemental intramuscular ketamine (10 mg/kg) and xylazine (0.6 mg/kg) as needed. Following anesthesia, a
24-gauge angiocatheter was inserted into a marginal ear vein to
facilitate antibiotic administration and a second catheter was inserted
into the central artery of the contralateral ear to obtain serum
samples. A solution of sparfloxacin (obtained from Rhone-Poulenc Rorer Pharmaceuticals, Inc., Collegeville, Pa.) for intravenous injection was
prepared with 5 ml of a 5% dextrose in water solution and 0.5 ml of
lactic acid (pH 3.6) and heated by means of a hot tap water bath. After
the sparfloxacin was dissolved, another 5 ml of 5% dextrose in water
was added to obtain a final concentration of 9.5 mg/ml. The solution
was administered by a rapid (1-min) intravenous infusion (40 mg/kg)
through a marginal ear vein, followed by a 1-ml flush with 0.9% NaCl.
Serial samples (blood and vitreous humor) were taken at 0.25, 0.5, 1, 2, 3, 4, 6, and 8 h after drug administration as previously
described (41). For the determination of sparfloxacin ocular
pharmacokinetics following systemic administration, six animals were
used. For direct injection studies with quinolone with and without
probenecid, 20 animals in four groups were used. Animals in each group
received either sparfloxacin, ofloxacin, ciprofloxacin, or fleroxacin;
one eye received both probenecid and a quinolone, and the other eye
received quinolone alone. For direct-injection experiments, solutions
of quinolones alone or in combination with probenecid or heat-killed
bacteria were injected into the midvitreous. Five additional animals
were used in the heat-killed-bacterium experiments. Probenecid was
dissolved in 1 N NaOH and adjusted to pH 8.6 prior to injection.
Probenecid was diluted in balanced salt to a final concentration of
2.86 µg/ml. The concentration of ciprofloxacin (Miles
Pharmaceutical Division, West Haven, Conn.), fleroxacin (Roche
Pharmaceuticals, Nutley, N.J.), ofloxacin (RWG Pharmaceutical
Research Institute, Raritan, N.J.), and sparfloxacin in the
direct-injection experiments was 5 µg/ml. Heat-killed S. epidermidis (ATCC 155) was prepared with an overnight inoculum
following three cycles of centrifugation and washing with 0.9% saline.
Thereafter, cells were spectrophotometrically adjusted to a final
inoculum of 109 with 0.9% saline and then heated to 80°C
for 20 min. One hundred microliters of 109 heat-killed
S. epidermidis organisms was injected via a 30-gauge needle
into the midvitreous cavity of one eye; the contralateral eye received
the same volume of 0.9% saline. For direct-injection experiments, 100 µl of each quinolone was injected into the midvitreous as previously
described (43). Following the designated sampling period,
animals were sacrificed with pentobarbital sodium solution (125 mg/kg)
and bilateral pneumothoraces.
Antibiotic assays.
To determine sparfloxacin concentrations
in the serum and vitreous, a well-diffusion microbiological assay was
used. Prior to analysis, all samples were stored at 
20°C. Blood
samples were allowed to clot and were immediately centrifuged at
1,000 × g for 15 min. The test organism was
Escherichia coli KL16. An inoculum of 107
organisms/ml diluted 1:10 in 3% brain heart infusion agar mixed with
Mueller-Hinton broth (Difco) adjusted to pH 8.0 with 1 N NaOH was used.
Wells (4-mm-diameter) were cut and 10-µl aliquots of serum or
vitreous humor were then pipetted into the wells. The agar was
incubated overnight at 37°C in an ambient-air incubator. Zones of
inhibition were read to the nearest 0.1 mm with a vernier caliper.
Sparfloxacin standards were prepared by dissolving 100 µg of drug per
ml in 1 mmol of NaOH per liter; this solution was then diluted with
either rabbit serum (for serum standards, 24, 12, 8, 4, and 2 µg/ml)
or balanced salt solution (for vitreous standards, 12, 6, 3, 1.5, 0.75, 0.375, and 0.1875 µg/ml). The sensitivity of the biological assay was
1.6 ng. The coefficients of variation in the biological assay for
the high and low standards were 4.3 to 7.5% and 0.4 to 3.1%,
respectively, with an assay linearity of 0.99. There is little or no
metabolism of sparfloxacin with no biologically active metabolites
(11, 30, 45, 50).
To compare the sensitivity of the biological assay to that of
high-pressure liquid chromatography (HPLC), sparfloxacin concentrations were also measured by HPLC according to the method of Borner et al.
(11). Samples were run at 25°C in a C18,
5-µm column (220 by 2.1 mm) packed with Nucleosil. Sample preparation
was performed by mixing 20 µl of serum with 130 µl of mobile phase
to acid precipitate proteins and by filtering. The mobile phase (75%
acetonitrile-25% 0.1 M H3PO4 adjusted to pH
3.82 with concentrated phosphoric acid) was delivered to the column at
a rate of 0.2 ml/min with a Hewlett-Packard (Wilmington, Del.) series
1050 pump. Serum samples were prepared in pooled rabbit serum. Vitreous
samples could not be assessed by HPLC because of the low sensitivity
(sparfloxacin does not fluoresce) of the assay. One hundred microliters
of sample was injected by a Hewlett-Packard series 1050 autosampler and
run serially through a Hewlett-Packard 1040A UV detector (240- to 280-nm wavelengths) and a Hewlett-Packard 1046A fluorescence detector (excitation, 280 nm; emission, 445 nm). Data were collected on a
Hewlett-Packard Chemstation. Quantitation of the antibiotic concentrations used peak heights. Antibiotic concentrations in the
serum and vitreous following systemic drug administration were
determined by HPLC (51); concentrations following direct injection were determined by the microbiological assay. The
coefficients of variation for the high and low standards were 2.4 and
2.2%, respectively.
Protein quantitation and characterization.
Protein
concentrations in the vitreous humor samples were determined with
Coomassie protein assay reagent (Pierce, Rockford, Ill.). The Coomassie
protein assay was performed by placing 1 µl of sample, 9 µl of
distilled water (dH2O), and 240 µl of Coomassie reagent
into each well of a 96-well microtiter plate. The plate was read on an
EL 312e Biokinetics Reader (Bio-Tek Instruments, Winooski, Vt.) at a
filter width of 630 nm. To prevent overloading of the sodium dodecyl
sulfate (SDS)-polyacrylamide gels, samples were diluted to a final
concentration of <4 µg/ml. Albumin standards (rabbit albumin; Sigma,
St. Louis, Mo.) were run at concentrations of 0.5, 1, 2, 4, 6, 8, and
10 µg/ml.
Identification and quantitation of proteins in the vitreous humor were
performed by SDS-polyacrylamide gel electrophoresis Mini-Protean II
cell, model with 1000/500 power supply; Bio-Rad, Hercules, Calif.) and
densitometry (model 60S video densitometer; BioImage, Ann Arbor,
Mich.). Minigels were run according to the method of Laemmli
(33). We used a 12% running gel, a 4.5% stacking gel, and
a Tris (0.25 M)-glycine (1.92 M)-SDS (1%) buffer. Samples were
prepared by using 1 µl of sample, 4 µl of dH2O, and 5 µl of sample solubilizer. Eight microliters of sample was loaded onto
the gel, which was run at 175 V for 40 to 45 min. The gel was stained
with Coomassie brilliant blue (J. T. Baker, Inc., Danvers, Mass.)
for 30 min and destained with a 5% acetic acid solution. Standards
included rabbit serum albumin (0.5, 2, and 4 µg/ml), rabbit lens
protein, and rabbit hemoglobin. Rabbit lens protein was obtained by
homogenizing surgically resected rabbit lenses after the capsules had
been removed. Rabbit hemoglobin was obtained from rabbit erythrocytes
that had been washed three times in phosphate-buffered saline (PBS) and
lysed in dH2O; cell fragments were removed by
centrifugation at 8,000 × g (Micro Centrifuge model
5415C; Brinkmann Instruments Inc., Westbury, N.Y.). An MW standard
(midrange kit; Enprotech, New York, N.Y.) and lens protein (diluted
40×) were also run with each gel. Albumin concentrations in vitreous
samples and sera were determined by densitometry.
Protein binding.
The protein binding was determined by
ultrafiltration of 4-ml standards at several concentrations of
sparfloxacin and other quinolones (1.0, 5.0, 10, and 20 µg/ml)
through Centriflo CF25 (MW cutoff, 25,000) membrane cones (Amicon, Inc.
Beverly, Mass.) according to the specifications of the manufacturer.
Standard solutions for each quinolone were prepared with rabbit serum
(Sigma). Briefly, cones were moistened with dH2O, placed
into their supports, and dried by centrifugation at 1,000 × g for 3 min. Ultrafiltration was performed at 780 × g for 10 min. Filter binding was determined by comparing
drug concentrations in ultrafiltrates prepared with PBS with those in
spiked PBS. Protein binding was adjusted to account for binding to the
filter. Concentrations of free drug in ultrafiltrates were determined
by the bioassay described above.
Lipophilicity.
The lipophilicities of the quinolones were
characterized by determining their partitioning ratios into octanol and
PBS by standard methods (15). Briefly, solutions containing
10 µg/ml in 0.1 M phosphate buffer (pH 7.2) were agitated with an
equal volume of n-octanol at 25°C for 48 h and
subsequently centrifuged at 1,870 × g for phase
separation. The concentrations of quinolones in the aqueous phase were
then determined by the microbiological assay. Partition coefficients
were expressed as the ratio of the amount of the compound in the
n-octanol phase to that in the aqueous phase.
Mathematical modeling and statistics.
Pharmacokinetic
analyses of the plasma and vitreous humor concentration-time
data following systemic administration were performed with RSTRIP
(Micromath Scientific Software, Salt Lake City, Utah), an iterative,
nonlinear, weighted, least-squares-regression program. The most
appropriate pharmacokinetic models were determined by using the
coefficient of determination and the RSTRIP model selection criterion,
which is a modified form of the Akaike (1) information criterion. Noncompartmental parameters were estimated by the
statistical-moment theory. Estimations for each exponential coefficient
and time constant were computed with the standard deviations of each
estimate, along with its 95% confidence range, which was calculated by
using both univariate and support-plane approximations for the bounds of the 95% confidence range. Other standard pharmacokinetic parameters were determined with computer-generated primary coefficients and standard pharmacokinetic equations (26, 27). Parameters were calculated for each animal; population pharmacokinetic parameters were
then calculated by a standard two-step technique (27).
To determine the relative contribution of MW, protein binding, and
hydrophobicity (independent variables) to the penetration
of quinolones
into the vitreous humor, we used multiple linear
regression (SYSTAT,
Evanston, Ill.). Penetration was expressed
as a percentage by dividing
the area under the concentration-time
curve (AUC) from 0 h to
infinity in the vitreous by that in the
serum following a single dose
of each quinolone. To determine
the relative importance of protein
binding, levels of penetration
were expressed as both total and free
fractions; the latter were
calculated as percent penetration (free) = percent penetration
(total) × (1% of protein bound). Since protein
concentrations
in the vitreous humor are less than 1% of those in
serum and since
animals with any breakdown of the blood-ocular barrier
(BOB) were
excluded from analysis, for these calculations we assumed
that
there was no binding in the vitreous humor. The logarithms of
the
mean penetration and of the mean free penetration were the
dependent
variables (
33,
50) in systemic-administration experiments.
In direct-injection experiments, the first-order elimination rate
half-lives were compared with the logarithm of the partition
coefficient
(
3). We performed univariate-linear-regression
analysis, employing
the octanol-water partition coefficient (the
permeability coefficient
[
p]), the square root of the MW,
the fraction of protein bound,
and a hybrid variable
(
p/
MW) as independent variables. (
46,
49) The
statistical significance of each of the variables was
determined
univariately (
6,
30,
46,
57). For the multiple
linear
regressions, each of the independent variables was allowed
to step in
(
P < 0.05) or step out (
P < 0.15).
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RESULTS |
Determination of sparfloxacin concentrations in serum and vitreous
humor.
Because of the small sample sizes (5 to 10 µl) used when
serial samples were obtained from the vitreous humor in our
ocular pharmacokinetic model (40, 41, 43), very sensitive
assay methods were required. As a result, we compared the
sensitivities and reproducibilities of results of HPLC and
microbiological assays for sparfloxacin using modifications of standard
assays previously described by others (11). The
sensitivities of the microbiological and HPLC assays were 1.9 and 25 ng, respectively. Thus, the biological assay was 14-fold more sensitive
than HPLC. For the biological assay, the coefficients of variation for
the high and low standards were 1 and 4.5%, respectively. No
metabolites were found in serum samples by HPLC.
Ocular pharmacokinetics of sparfloxacin.
Data from six
animals with no breakdown of the blood-vitreous barrier, as
determined by SDS-polyacrylamide gel electrophoresis, were analyzed
(Fig. 1). Results are plotted
arithmetically and semilogarithmically to better demonstrate the
relative levels of penetration and terminal elimination slopes,
respectively. Model-predicted and actually observed drug concentrations
in the serum and vitreous were similar (Table
1). Both hybrid and derived microconstants are given in Table 2.
Model-dependent analysis gave excellent fit with coefficients of
determination for the serum and vitreous of 0.999 and 0.997, respectively. The AUCs in the vitreous humor and serum were 14.43 and
22.03 mg · h/liter, respectively. Penetration into the vitreous
humor was 59.4% ± 12.2% of that in the serum. The terminal
elimination rate constants in the vitreous humor and serum were 0.28 and 0.24, respectively. The elimination half-life in the vitreous humor
was 2.99 h, and that in serum was 2.39 h (P > 0.05). On the basis of the coefficient of determination and model
selection criterion, vitreous humor and serum antibiotic
concentration-time data following intravenous administration were
best-fitted to a two-compartment model.
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FIG. 1.
Mean concentrations of sparfloxacin in the serum and
vitreous humor of six rabbits following a single intravenous dose (40 mg/kg). The left graph shows the data plotted arithmetically, and the
right graph shows the data plotted semilogarithmically.
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TABLE 1.
Comparison of measured and
pharmacokinetic-model-predicted sparfloxacin concentrations in serum
and vitreous humor following systemic administration
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Correlation between physicochemical properties and protein binding
and ocular translocation.
The second goal of this ocular
pharmacokinetic study was to determine the relationship of
lipophilicity, MW, and protein binding to translocation across the
blood-ocular barrier of the quinolone antimicrobial following systemic
and direct injections. The translocation of sparfloxacin was compared
to those of three other quinolones (ciprofloxacin, fleroxacin, and
ofloxacin) for which we have previously shown significant differences
in levels of ocular penetration (Fig. 2)
(23, 41, 43, 51). Among the four quinolones studied, levels
of penetration differed by an order of magnitude; levels of
ciprofloxacin and sparfloxacin penetration were 5.5 and 59%, respectively. The effects of three independent variables on ocular penetration were considered in the multiple-linear-regression model:
lipophilicity, MW, and protein binding.
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FIG. 2.
(A) Relationship between the partition coefficients for
ciprofloxacin ( ), fleroxacin ( ), ofloxacin ( ), and
sparfloxacin ( ) and levels of penetration into the vitreous humor.
(B) Relationship between the partition coefficients for these
quinolones (same symbols) and the elimination rate half-lives following
direct intravitreal injection.
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Table
3 shows the ocular penetration of
each drug along with its MW, level of protein binding, and partition
coefficient.
Only the lipophilicities were statistically significant
when examined
univariately. This relationship is described by the
equation log(mean
percent vitreal penetration) = 2.739(
p) + 0.59, where
p is the
octanol-water partition coefficient
(
r2 > 0.999,
P < 0.001). Multiple linear regression was then undertaken
after
considering additional variables, including MW and the free
fraction of
drug in the serum available for transport; these additional
variables
did not improve model fit.
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TABLE 3.
Relationship of lipophilicity, protein binding, and MW to
the penetration of four quinolones into the vitreous humor
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To determine if carrier-independent translocation across the
blood-ocular barrier following direct injection also correlated
with
the physicochemical properties of quinolones, we also determined
the association between lipophilicity and drug elimination following
direct injection into the vitreous humor in 20 animals. One eye
received the quinolone alone, and the other eye received both
the
quinolone and probenecid. The elimination half-lives for ciprofloxacin,
fleroxacin, ofloxacin, and sparfloxacin were 4.41, 3.35, 3.04,
and
2.78 h, respectively (Table
4). As
with systemic-injection
experiments, there was an excellent correlation
between lipophilicity
and efflux (
r2 > 0.99,
P < 0.01); MW and the free fraction did not
improve model
fit. The relationship is described by the equation
t1/2
=
(
1.8172)(log
10p) + 2.1239, where
t1/2 is the half-life at
beta phase.
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TABLE 4.
Comparison of the effects of probenecid on the
half-lives for elimination from the vitreous humor of
quinoline antimicrobials
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Effects of probenecid and heat-killed bacteria on quinolone
elimination following direct injection.
Since the renal
elimination of quinolones and beta-lactam antibiotics in humans and
rabbits is blocked by probenecid and since the ocular elimination of
the carrier-mediated export of beta-lactams from the vitreous humor is
blocked by both probenecid and heat-killed bacteria (8, 25,
37), we examined the effects of each on the elimination of
quinolones following direct injection. As shown in Fig.
3 and Table 4, probenecid significantly
increased the elimination half-lives of ciprofloxacin, fleroxacin, and
sparfloxacin (P < 0.05). While probenecid also
increased the elimination half-life of ofloxacin (4.15 h with
probenecid versus 3.04 h without), this difference was not
significant (P = 0.15). Heat-killed bacteria also
increased the elimination half-life of fleroxacin 1.42-fold (P < 0.01). The effects of inflammation on the
elimination rates of ciprofloxacin, ofloxacin, and sparfloxacin were
not tested.
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FIG. 3.
Effect of probenecid on the elimination of sparfloxacin
(A), fleroxacin (B), ofloxacin (C), and ciprofloxacin (D) with ()
and without () the coadministration of probenecid; both probenecid
and the quinolones were given intravitreally. The inset shows the
effect of heat-killed S. epidermidis () on the
elimination of fleroxacin alone ().
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DISCUSSION |
Because of the small sample sizes obtained from the vitreous
humor, very sensitive assay methods were required. When we compared the
sensitivities of HPLC and microbiological assays for sparfloxacin, the
latter method proved to be 14-fold more sensitive than HPLC with
coefficients of variation for the high and low standards of 1 and
4.5%, respectively. No metabolites were found in serum samples by
HPLC. Recent studies in our laboratory with the quinolone ciprofloxacin
have shown that, in general, the sensitivities and reproducibilities of
results of HPLC and biological assays are equivalent. However, the
activities of quinolones differ in the presence and absence of
microbiologically active metabolites and quinolones differ in their
capacities to fluoresce. For compounds with active metabolites
(50) (e.g., ciprofloxacin and ofloxacin), HPLC is the
preferred assay method when drugs are administered systemically. On the
other hand, for compounds like fleroxacin, for which there are no
active metabolites (57), the biological assay is preferred
(43). Like fleroxacin, sparfloxacin differs from
ciprofloxacin and ofloxacin by not having biologically active metabolites. However, unlike other quinolones, sparfloxacin does not
fluoresce; the sensitivity of HPLC assays with quinolones is increased
by at least an order of magnitude when fluorescent compounds are used.
As a result, when doses of sparfloxacin that mimic those achieved in
the sera of humans were used, the HPLC assay was not sufficiently
sensitive to measure drug concentrations in ocular fluid.
Sparfloxacin showed excellent penetration into the vitreous
humor, with mean concentrations in the vitreous humor of
uninflamed eyes of 59.4% ± 12.2% of that in the serum. Following
systemic administration, the elimination half-life from the
vitreous in rabbits was 3.34 h and that from the serum was
2.2 h. The terminal-elimination half-life and maximum
concentration of sparfloxacin in human serum were 17.6 h and 1.6 µg/ml, respectively. (30) The maximum concentrations in
the serum and vitreous of rabbits following a 40-mg/kg bolus, achieved
at approximately 1 hour after intravenous administration, were 12.43 and 2.84 µg/ml, respectively. While albino rabbits were used in this
study, previous experiments in our laboratory have shown that the
levels of penetration of other quinolones, namely, ofloxacin and
ciprofloxacin, into the vitreous humor are identical in pigmented and
nonpigmented animals (51).
Recent pharmacokinetic studies by Cochereau-Massin and colleagues
with pigmented, uninfected rabbits showed the maximum vitreal concentration to be 5.6 µg/ml, with a level of penetration of 54%
following systemic injection of 50 mg/kg (16). Those authors also showed that sparfloxacin was more efficacious in the therapy of
staphylococcal endophthalmitis in rabbits than
systemically administered vancomycin or amikacin (37).
Importantly, newer quinolones such as sparfloxacin (35) and
ofloxacin (51) not only show better penetration into the
vitreous humor than other quinolones such as ciprofloxacin
(P < 0.05) but also are more active against
gram-positive bacteria commonly isolated from patients with
endophthalmitis.
Using multiple linear regression, we have shown that there was an
excellent correlation between lipophilicity and penetration into or
elimination from the eye following systemic or intravitreal injection in nonpigmented, uninfected rabbits. Both lipophilicity and
protein binding were measured under conditions that mimic those in
ocular tissue and serum; lipophilicity was measured at physiological pH, and protein binding was measured with rabbit sera. The penetration of quinolones across the outer lipid
membrane of E. coli is also proportional to
lipophilicity. (28) Additionally, studies using artificial
lipid membranes show that lipophilicity rather than molecular size best
correlates with penetration (49).
Carrier-independent penetration of antibiotics and other drugs into the
eye (10, 13, 19, 44, 53, 61), like that at other anatomical
sites (3, 13, 19, 30, 44, 46, 53, 61), correlates with
physicochemical properties, including lipophilicity and MW as well as
protein binding. These independent variables were considered in the
multiple-linear-regression model. The log of the penetration of
compounds across planar lipid bilayers and tissue correlates with the
oil/water partition ratio, the inverse of the square route of the MW
(17, 20, 46, 50), and the free fraction of drug (44,
62). As shown in our studies, lipophilicity is generally the most
important variable.
Our inability to discern the potential role of protein binding was
limited because of the restricted range examined for this independent
variable. Since protein binding clearly affects antibiotic penetration
into tissue (30, 44, 46), studies with quinolones which show
greater differences in these properties are planned. It has been
difficult to establish rigorous models characterizing the importance of
these properties in antibiotic penetration with single samples
(56) which are pooled from different animals (10, 13,
19, 44, 53, 61).
While we showed an excellent correlation between lipophilicity
and translocation, modeling of quinolone elimination rates following systemic injection compared to that following direct injection suggested that drug elimination from the eye was more rapid
than drug entry. This finding, in conjunction with results of studies
showing that the renal and ocular eliminations of both quinolones and
beta-lactam antibiotics in humans and rabbits are blocked by probenecid
(4, 5, 9, 12, 18, 28) and inflammation (8, 25),
suggested that more than one mechanism may be involved in
elimination from the vitreous humor. To test this hypothesis, the
elimination rates of four quinolones were examined following direct
injection. Both probenecid and heat-killed bacteria prolonged the
elimination rates of quinolones following direct injection. These
observations suggest that the elimination of quinolones from the eye
likely involves carrier-independent translocation via biological
membranes with tight capillary and/or retinal cell barriers as well as
carrier-dependent elimination blocked by both probenecid and
heat-killed bacteria. In vivo and in vitro studies characterizing the
rates of renal elimination of zwitterionic quinolones suggest the
presence of separate and distinct carrier-mediated systems (29,
47). As a result, while the experiments with probenecid and
heat-killed organisms suggest carrier-mediated export from the eye, the
nature of this carrier(s) is unknown.
The excellent correlation between lipophilicity and penetration or
elimination also demonstrates the strength of our animal model in
comparison to that in which one sample obtained from different animals
is pooled to generate single-subject estimates. In addition to
characterizing the effects of physicochemical properties on ocular
pharmacokinetics, using this animal model we have characterized the effects of different modes of drug administration on ocular penetration (35), established a model that permits the
determination of robust ocular pharmacokinetic parameters in the
serum and vitreous humor using sparse datum sets (47), and
described the ocular pharmacokinetics of quinolones following direct
and systemic drug administration in pigmented and albino rabbits
(23, 49). These experiments would have been difficult if we
had used the single-sample methods employed in most ocular
pharmacokinetic studies of animals.
We have developed mathematical models that are highly explanatory of
the penetration of and elimination from the vitreous of
fluoroquinolones which differ mostly in their octanol-water partition
coefficients. We have also shown that quinolones, like the beta-lactam
antibiotics, are exported from the vitreous humor via a pump which is
blocked by both probenecid and inflammation. It will be important in
future studies to examine accuracy of prediction of penetration into
the vitreous for additional quinolones as well as other drugs whose
efficacies are dependent upon penetration into the vitreous humor.
This will allow this model either to be further validated or to
be shown to be only locally predictive for fluoroquinolones, in which
case, other independent variables may be needed to make the model
more globally predictive.
| |
ACKNOWLEDGMENTS |
This work was supported by grant RO1EY089977-01 from the National
Eye Institute and an unrestricted grant from Research to Prevent
Blindness.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Medicine, Albany Medical College, 47 New Scotland Ave., Albany, NY
12208. Phone: (518) 262-5343. Fax: (518) 262-6727. E-mail:
michael_miller{at}ccgateway.amc.edu.
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Abstract
Introduction
